In silico cloning - Bits.vib.be

Tutorial 1: Primer design
In this tutorial we will use the CLC Main Workbench to design primers for PCR amplification of a
specific region in the DNA. We will use the pcDNA3-atp8a1 sequence from the 'Primers' folder in
the Example Data. This is the pcDNA3 vector with the atp8a1 gene inserted. We wish to design
primers that allow us to generate a PCR product covering the insertion point of the gene to check if
the gene is inserted where we think it is.
First, open the sequence and then open the Primer Designer
Now the sequence is opened and we are ready to begin designing primers.
1.1 Specifying a region for the forward primer
First Zoom out to get a single-line overview of the sequence by clicking Fit Width.
We want the forward primer in a region between position 600 and 900 – just before the gene (use
the Find settings in the Side Panel to make the selection).
Select this region, right-click it and choose "Forward primer region here"

This will add an annotation to this region, Forward primer region, (unselect Show in the Restriction
sites settings in the Side Panel to get a more clear view of the annotations) and 5 rows of red and
green dots (you have to zoom in to see the dots: use the 100% button) representing the primer
suggestions. There is a line for each primer length - 18bp through to 22 bp, hence five lines.
1.2 Examining the primer suggestions
Each line consists of a number of dots, each representing the starting point of a possible primer,
e.g. the first dot on the first line represents a primer starting at the dot's position and with a length
of 18 nucleotides. The primer will anneal to the selected region in the DNA.
Position the mouse cursor over a dot. A box will appear, providing data about this primer.

Clicking the dot will select the region where that primer would anneal. Note that some of the dots
are colored red. This indicates that the primer represented by this dot does not meet the
requirements set in the Primer parameters that you can see in the Side panel on the right:
The default maximum melting temperature is 58. This is the reason why the primer in the figure
below with a melting temperature of 58.55 does not meet the requirements and is colored red.
There is an asterisk (*) before the melting temperature. This indicates that this primer does not
meet the requirements regarding melting temperature. In this way, you can easily see why a
specific primer fails to meet the requirements.

If you raise the maximum melting temperature to 59, the primer will meet the requirements and the
dot becomes green.
Below you see that both melting temperature and GC content do not meet the requirements.

By adjusting the Primer parameters in the Side Panel you can define primers to meet your needs.
Since the dots are dynamically updated, you can immediately see how a change in the primer
parameters affects the number of red and green dots.
Remember: each dot represents the starting point of a primer: you must look for individual green
dots: they represent primers that meet the requirements. You do not need to look for 18
consecutive green dots: one dot represents a whole primer.
1.3 Calculating a primer pair
Until now, we have been looking only at the forward primer. To mark a region for the reverse primer,
make a selection from position 1200 to 1400 and:
Right-click the selection | Reverse primer region here

Now, you can let CLC Main Workbench calculate all the possible primer pairs based on the Primer
parameters that you have defined:
Click the Calculate button in the Primer Designer Settings
This opens the Calculation parameters window:
You can modify the parameters of the combination of primers. If you leave them unchanged and
you just click 'Calculate' a table is opened showing the possible combinations of primers.

In the Side Panel of the view containing the primer pairs you can adjust the Primer Table Settings,
to specify the info you want to display in the table, e.g. Fragment length
Clicking a primer pair in the table will make a corresponding selection on the sequence in the view.
At this point, you can settle on a specific primer pair or save the table for later. If you want to use
the first primer pair for your experiment, right-click the primer pair in the table and save the primers.

You can also mark the position of the primers on the sequence by selecting Mark primer annotation
on sequence in the right-click menu.
This results in two annotations on the sequence in the view.

Tutorial 2: In silico cloning workflow
In this chapter, the goal is to virtually PCR-amplify a gene using primers with restriction sites at the
5' ends, and to insert the gene into a multiple cloning site of an expression vector. The vector acts
as a carrier for your gene of interest. To ensure that your gene of interest is inserted in the correct
orientation we will use two different restriction enzymes, creating different ends. The multiple
cloning site of the vector contains numerous restriction sites allowing us to choose two enzymes
that create different ends and that do not cut in our gene of interest. We start off with a set of
primers, a DNA template sequence and an expression vector loaded into the Workbench.
2.1 Locating the data to use
Open the Example data folder in the Navigation Area. Open the Cloning folder, and inside this
folder, open the Primers folder.
Double-click the ATP8a1 mRNA sequence in the Example Data folder and zoom to Fit Width and
you will see the light green annotation which is the coding part of the gene. This is the part that we
want to insert into the pcDNA4_TO vector in the Cloning folder. The primers have already been
designed using the primer design tool.
2.2 Add restriction sites to primers
First, we add restriction sites to the primers. In order to see which restriction enzymes can be used,
we create a split view of the vector and the fragment to insert. In this way we can easily make a
visual check to find two enzymes from the multiple cloning site in the vector that do not cut in the
gene of interest. To create the split view:
double-click the pcDNA4_TO sequence | View | Split Horizontally
For the vector sequence switch to the Circular view.
Zoom in on the multiple cloning site downstream of the green CMV promoter annotation. You
should now have a view similar to this:

By looking at the enzymes we can see that HindIII and XhoI cut in the multi-cloning site of the vector
and not in the Atp8a1 gene. You can see the ends that are created by these enzymes by going
to the Restriction Sites group in the Side Panel. Select ‘Sort enzymes by overhang type’ ( )
in Sorting and place your cursor over HindIII in the Side Panel. You will see how the enzyme cuts.
Note that you can add more enzymes to the list in the Side Panel by clicking Manage Enzymes
under the Restriction Sites group.

You can do all kinds of restriction analysis. Suppose you want to know which restriction enzymes
will cut the ATP8a1 mRNA sequence exactly once and create a 3’ overhang. Do the following:
Select the ATP8a1 mRNA sequence | Toolbox in the Menu Bar |
Cloning and Restriction Sites | Restriction Site Analysis |
Select ATP8a1 mRNA sequence | Click Next

Select Use existing enzyme list | Click the Browse for enzyme list button ( ) |
Select ’Popular enzymes’ in the Cloning folder under Enzyme lists | click OK
Write 3’ into the left filter | Select all enzymes | Click the Add (
) button

Click Next
In this step you specify that you want to use enzymes that cut the sequence only once.
Deselect the Two restriction sites checkbox | click Next
Select Add restriction sites as annotations on sequence | Create restriction map | Finish
You see that there are only two enzymes that fulfill these criteria (SacII and KpnI).

We come back to our in silico cloning problem. If you have chosen the enzymes that you want to
work with (in our case: HindIII and XhoI), close the views of the vector and the mRNA sequence
and open the ATP8a1 fwd primer sequence. When it opens, double-click the name of the sequence
to make a Selection of the full sequence. If you do not see the whole sequence turn
purple, please make sure you have the Selection Tool chosen.
If the sequence is selected, right-click and choose Insert Restriction Site Before Selection
In the Filter box enter HindIII and click on it. At the bottom of the dialog, add a few extra bases 5'
of the cut site (this is done to increase the efficiency of the enzyme).

Click OK and the sequence will be inserted at the 5' end of the primer.
Perform the same process for the ATP8a1 rev primer, this time using XhoI instead. This time, you
should also add a few bases at the 5' end as was done when inserting the HindIII site to increase
to efficiency of the enzyme.
Note! The ATP8a1 rev primer is designed to match the negative strand, so the restriction site
should be added at the 5' end of this sequence as well (using Insert Restriction Site before Selection).
Save the two primers and close the views.
2.3 Simulate PCR to create the fragment
Now, we want to extract the PCR product from the template ATP8a1 mRNA sequence using the
two primers with restriction sites:
Toolbox | Primers and Probes | Find Binding Sites and Create Fragments

Select the ATP8a1 mRNA sequence | Click Next
Use the Browse button to select the two primer sequences | Click OK
Leave the other parameters at their default values.
The Match criteria determine how well a primer should match the sequence. Exact match specifies
that all positions of the primer must base pair with the sequence. Note that we cannot choose
this option because we have added non-matching restriction sites to the primers. We have to de-

fine the minimum number of positions and the number of consecutive positions in the 3’ end
of the primer that must base pair to the sequence in order to speak of a match.
Note that the number of mismatches is reported in the output, so you can filter on this afterwards.
Below the Match criteria, you can adjust Concentrations concerning the reaction mixture. This is
used when reporting melting temperatures for the primers.
Click Next | Adjust the output options
Click Finish
You now see the fragment table displaying the PCR product. In the Side Panel you can choose to
show information about melting temperature for the primers.
Right-click the fragment and select Open Fragment.

This will create a new sequence representing the PCR product. Save the sequence in the Cloning
folder and close the views. You do not need to save the fragment table.
2.4 Specify restriction sites and perform cloning
The final step is to insert the fragment into the cloning vector:
Toolbox in the Menu Bar | Cloning and Restriction Sites | Cloning
Select the Fragment (ATP8a1 mRNA (ATP8a1 fwd - ATP8a1 rev)) sequence | click Next

Use the Browse button to select pcDNA4_TO cloning vector in the Cloning folder | click OK
Click Finish
You now see the cloning editor with the pcDNA4_TO vector in a circular view. Press and hold the
Ctrl key while you click first the HindIII site and next the XhoI site.

At the bottom of the view you can now see information about how the vector will be cut open. Since
the vector has now been split into two fragments, you can decide which one to use as the target
vector. If you selected first the HindIII site and next the XhoI site, the CLC Main Workbench has
already selected the right fragment as the target vector. If you click one of the vector fragments,
the corresponding part of the sequence will be highlighted.

Next step is to cut the fragment. At the top of the view you can switch between the sequences
used for cloning (at this point it says pcDNA4_TO 5.078bp circular vector).
Switch to the fragment sequence and perform the same selection of cut sites as before while
pressing the Ctrl key.
At the bottom of the view you can again see information about how the fragment will be cut. Since
the fragment can split into two fragments, you can decide which one to use as the target fragment.
If you selected first the HindIII site and next the XhoI site, the CLC Main Workbench has already
selected the right fragment. If you click one of the fragments, the corresponding part of the sequence
will be highlighted.

When this is done, the Perform Cloning (
) button at the lower right corner of the
view is active because there is now a valid selection of both fragment and target vector.
Click the Perform Cloning button.
This dialog lets you inspect the overhangs of the cut site, showing the vector sequence on each
side and the fragment in the middle. The fragment can be reverse complemented by clicking the
Reverse complement fragment (
your new construct will be opened.
) but this is not necessary in this case. Click Finish and
When saving your work, there are two options:
Save the Cloning Experiment. This is saved as a sequence list, including the specified cut
sites. This is useful if you need to perform the same process again or double-check details.

Save the construct shown in the circular view. This will only save the information on the
particular sequence including details about how it was created (this can be shown in the
History view).
You can, of course, save both. In that case, the history of the construct will point to the sequence
list in its own history.
2.5 Perform cloning using one restriction site in the vector
Suppose you have a system that allows you to insert your fragment in the vector by just cutting the
vector once.
We will take the same vector as in the previous example pcDNA4-T0 and insert the full
atp8a1mRNA sequence. Since this fragment has blunt ends we will cut the vector using a blunt
end cutter. To find a cutter open the vector sequence in circular view, go to the Restriction sites
area in the Side Panel and click Manage Enzymes.
This opens the Manage enzymes window that allows you to select the restriction sites that you
want to display on the view. Filter on “blunt” and select the 4 most popular blunt end cutters.
Click Finish. In the same area on the Side Panel select Single cutters and deselect all others.

You see that you can use EcoRV to linearize the vector.
Toolbox | Coning and Restriction Sites | Cloning
Select atp8a1mRNA. Click Next. Select vector pcDNA4-T0. Click Finish. Select EcoRV restriction
site.

Switch to the fragment sequence. Click Add as fragment. Click Clone. Click Finish.
The vector now contains the complete atp8a1mRNA fragment. You see that the process for
working with a signle restriction site is exactly the same as for two restriction sites.

Tutorial 3: Batch processing
Most of the analyses in the Toolbox are able to perform the same analysis on several elements in
one batch. This means that analyzing large amounts of data is easily accomplished. As an example,
if you use the Find Binding Sites and Create Fragments tool and you supply five sequences,
the result table will present an overview of the results for all five sequences.
If you want individual outputs for each sequence, you would need to run the tool five times, or alternatively
use the Batching mode. Batching mode is activated by clicking the Batch checkbox in
dialog where the input data is selected.
Batching simply means that each data set is run separately, just as if the tool has been run manually
for each one. When batching is selected, the data to be added is the folder containing the data
you want to batch. When the folder is selected and you click Next, a batch overview is shown.
When you click Next the subsequent dialog allows you to specify the parameters of the Find Binding
Sites and Create Fragments analysis just as in a regular non-batch analysis.

Note that the Workbench is validating a lot of the input and parameters when running in regular
non-batch mode. When running in batch, this validation is not performed, and this means that
some analyses will fail if combinations of input data and parameters are not right. Therefore batching
should only be used when the batch units are very homogenous in terms of the type and size of
data.
At the last dialog before clicking Finish, it is only possible to use the Save option. When a tool is
run in batch mode, it will place the result files in the same folder as the input files.

Tutorial 4: Gateway cloning
The Workbench allows to perform in silico Gateway cloning, including Multisite Gateway cloning.
In the Gateway cloning system your gene of interest is inserted into an entry vector. The entry vector
contains attP recombination sites. To insert your gene of interest into the entry vector, you need
to create a PCR product with terminal attB sites, using primers containing a 25-base pair attB sequence,
plus four terminal G's. This PCR product can then be inserted in the entry vector using the
BP reaction, resulting in the formation of an entry clone (purple vector at the top) carrying the gene
of interst flanked by attL sites. Once the entry clone has been formed it is available to express your
gene in a variety of other destination vectors (red vector on the top) which contain attR sites using
an LR reaction. The result is an expression clone (red vector at the bottom) with your gene of interest
inserted in the destination vector.
The attB’s, attP’s, attL’s and attR’s are the key component of the Gateway system: they are the
restriction sites. All entry clones have attL's on both sides of their gene of interest. These attL's are
cut to form sticky ends by the Gateway recombination proteins. These sticky ends will then match
up with the sticky ends on a destination vector which contains attR restriction sites. This process is
called a LR reaction. The BP reaction is done similarly using other restriction sites. Note that the
LR reaction creates attB sites again in the expression clone: this makes the Gateway system totally
reversible. You can make more entry vectors from the expression clone using a BP reaction, in
which the attB sites recombine with attP's to form attL's and attR's.
The system has a high transformation efficiency since it uses both antibiotic resistance and the
ccdB gene, which yields a product that is toxic to E. coli, to ensure that only correct clones survive.
The three tools for Gateway cloning mimic the procedure followed in the lab:
1. attB sites are added to a sequence fragment
2. attB-flanked fragment is recombined in an entry vector (BP reaction) to construct an entry clone
3. target fragment from the entry clone is recombined into a destination vector (LR reaction) to
construct an expression clone.
For Multisite Gateway cloning, multiple entry clones can be created that can recombine in the LR
reaction. To perform these analyses in the CLC Main Workbench, you need to import donor and
expression vectors. These can be downloaded from Invitrogen’s web site and directly imported into
the Workbench: http://igene.invitrogen.com/products/selector/vectors.

4.1 Add attB sites
The first step is to amplify the target sequence with primers including the so-called attB sites. Suppose
you want to clone the CDS of ATP8a1. First you open the ATP8a1 mRNA sequence in the
Main Area. In the Sequence Settings tab of the Side Panel open the Annotation types settings and
deselect Gene.
Select the CDS. Right click the CDs and choose Open Annotation in New View.
Save the CDS as ATP8a1 CDS and add attB sites to the ATP8a1 CDS sequence fragment in this
way:
Toolbox | Cloning and Restriction Sites | Gateway Cloning | Add attB Sites

This will open a dialog where you can select on or more sequences.
When you have selected your fragment(s), click Next.

This will allow you to choose which attB sites you wish to add to each end of the fragment. If you
have selected several fragments and wish to add different combinations of sites, you will have to
run this tool once for each combination.
Clicking Next allows you to extend the fragment with additional sequences by extending the primers
5’ of the template-specific part of the primer (between the template specific part and the attB
site). For example you can add a Shine-Dalgarno site between the attB and the gene of interest.
Press Shift – F1 and you will see a list of possible additions from which you can choose.
This list shows you the most common additions, but of course you can manually type or paste
whatever sequence that you want to add. Use the up and down arrow keys to select a tag and
press Enter. This will insert the selected sequence in the primer.
At the bottom of the dialog, you can see a preview of what the final PCR product looks like. In the
middle is the sequence of interest (i.e. the sequence you selected as input). In the beginning is the
attB1 site and at the end the attB2 site. The primer additions you have inserted are shown in color.
Note that the four terminal G’s are automatically added to the attB sites.
Clicking Next allows you to specify the length of the template-specific part of the primers.

The Workbench is not doing any primer design. You specify the length of the part of the primers
that should base pair with the ATP8a1 CDS, together with the attB sites and optional primer additions,
and these are the primers. The Workbench will not check the compatibility of the primers, the
annealing temperatures or the GC-content.
Click Next.
Click Finish.
The main output is the input sequence now including attB sites and primer additions. The attB sites,
the primer additions and the primer regions are annotated in the final result. There will be one output
sequence for each sequence you have selected for adding attB sites.

You also get a list of primers. Again deselect Gene from Annotation types settings.
Save the resulting sequence as it will be the input to the next part of the Gateway cloning workflow.
When you open the sequence again, you may need to switch on the relevant annotation types to
show the sites and primer additions.
4.2 Create entry clones (BP)
The next step in the Gateway cloning workflow is to recombine the attB-flanked sequence of interest
into an entry vector to create an entry clone, the so-called BP reaction. First, you have to download
the entry vector (pDONR221) from Invitrogen’s web site (in the MultiSite Gateway® Three-
Fragment Vector Construction Kit; http://igene.invitrogen.com/products/selector/vectors) and import
it into the CLC Main Workbench if it’s not yet present in the Cloning vector library folder in the
Cloning folder in the Navigation Area.
Expand the Cloning vector library folder and you see pDONR221 at the bottom of the list.

Make sure that you circularize the vector and save it before you proceed with the actual cloning.
Toolbox | Cloning and Restriction Sites | Gateway Cloning | Create Entry Clone
Now you can select one or more sequences that will be the sequence of interest to be recombined
into your entry vector. Note that the sequences you select should be flanked with attB sites. You
can select more than one sequence as input, and the corresponding number of entry clones will be
created. When you have selected your sequence(s), click Next.

Next you have to specify the entry or donor vector that you are going to use.
Clicking the Browse button opens a dialog where you can select a donor vector.
Click Next, choose to Open the results and click Finish.

The Workbench looks for the attP sites in the sequences of the entry vector that you select in this
dialog so you should make sure that the attP sites are present in the entry vector that you choose.
It only checks that valid attP sites are found - it does not check that they correspond to the attB
sites of the selected fragments at this step. If the right combination of attB and attP sites is not
found, no entry clones will be produced.
The output is one entry clone per sequence selected. The attB and attP sites have been used for
the recombination, and the entry clone is now equipped with attL sites. The by-product of the BP
recombination is not part of the output. Save the results since you will need them for the subsequent
LR reaction.
4.3 Create expression clones (LR)
The final step in the Gateway cloning workflow is to recombine the entry clone into a destination
vector to create an expression clone, the so-called LR reaction:
Toolbox | Cloning and Restriction Sites | Gateway Cloning | Create Expression Clone
This will open a dialog where you can select on or more entry clones. If you wish to perform separate
LR reactions with multiple entry clones, you should run the Create Expression Clone in
batch mode.

When you have selected your entry clone(s), click Next.
Now you can select a destination vector. You can download destination vectors from Invitrogen’s
web site: http://igene.invitrogen.com/products/selector/vectors and import them into the Workbench.
Follow exactly the same procedure as for the entry vector. Don’t forget to circularize the
vector and save it.
The Workbench looks for the specific sequences of the attR sites in the sequences that you select
in this dialog. The Workbench only checks that valid attR sites are found - it does not check that
they correspond to the attL sites of the selected fragments at this step. If the right combination of
attL and attR sites is not found, no entry clones will be produced.

When performing multi-site Gateway cloning, the CLC Main Workbench will insert the fragments
(contained in entry clones) by matching the sites that are compatible. If the sites have been defined
correctly, an expression clone containing all the fragments will be created.
Click Next if you wish to adjust how to handle the results. If not, click Finish.
The output is a number of expression clones depending on how many entry clones and destination
vectors that you selected. The attL and attR sites have been used for the recombination, and the
expression clone is now equipped with attB sites. You can choose to create a sequence list with
the by-products as well.